To examine the structure of biological samples such as tissues (histology) or cells (cytology), microscopical preparations are made by mounting the sample on a substrate such as a microscope slide. These preparations are routinely stained with dyes to facilitate microscopical examination. To further aid in the identification of the samples, specialized procedures under the general headings of histochemistry (tissue slices) and cytochemistry (biological cell smears) are applied to these preparations. One class of procedures for processing biological materials involves ligand-pair formation wherein a first member of the ligand pair may be present in the biological sample and the other member of the pair binds to the first member when contacted with the sample. Examples of such biologically based ligand-pairs include antibody/antigen couples, lectins/sugars, hormone/receptor systems, enzyme/substrates, DNA/DNA and DNA/RNA couples.
Processing of biological materials involving the antibody/antigen couple forms the basis of immunohistochemistry and immunocytochemistry. Until recently the staining of mounted samples using these reactions has been performed manually. Present machines currently known for immuno-staining of samples dispense the antibody containing solutions in liquid form into the fluid well containing the supported sample. These machines require considerable operator attention which entails high labour costs and are prone to suffer from operator error at the stages of dilution, pipetting and loading of reagents. Furthermore, in many circumstances it may be desirable to detect different antigens on an ad hoc basis but the primary antibodies are expensive and prone to deterioration. In addition, the difficulties of working with a large number of small volumes used for a multitude of different tests has acted as a bar to the development of an optimally automated immunocytochemistry staining system.
There are several known methods for storing ligands temporarily. U.S. Pat. No. 5,068,198 issued to Gibbons teaches a temporary confining agent for one member of a specific binding pair (sbp) which is confined in a material to prevent it from combining with the other complementary member of the specific binding pair. Gibbons teaches-the reversible confinement means as being finely divided or particulate, specifically for use in a single liquid medium or reagent wherein the confinement means is suspended to provide for rapid release of the confined sbp member and consequent binding with the nonconfined complementary sbp member present in the liquid medium. The particles may be spherical or irregularly shaped, and normally have average diameters of 10 nm to 500 nm, more usually 20 nm to 2 .mu., frequently 100 nm to 1 .mu.. The confinement materials are compatible with and insoluble in the liquid medium, usually an aqueous buffer solution, and are comprised of "immiscible" materials. The principal example taught by Gibbons is liposomes formed such that the outer surface of the liposomes are "substantially free of sbp members." Thus, the confinement means forms a barrier between one member of an sbp enclosed therein and the complementary member of the sbp present in the liquid medium in which the particulate confinement means is suspended.
Although Gibbons' teachings include gels like gelatin as example confinement means, those skilled in the art would recognize that water soluble gels, like gelatin, would be miscible not "immisible" with aqueous buffers. Also, a simple mixture of a water-soluble ligand and a water-soluble confinement means, or other mixtures of ligands and confinement means both soluble in a common solvent, would when formed into particles express the ligand on the particle surface.
U.S. Pat. No. 5,413,924 issued to Kosak is directed to a method of entrapment of liquid reagents such as enzymes or antibodies in wax beads, granules, liposomes and the like which are released upon heating into the surrounding medium such as a solution. The Kosak patent is directed to waxy or wax-like polymers that are essentially water insoluble materials that are solid or semi-solid at room temperature. Although heat-releasable gels, including gelatins are cited as entrapment means, Kosak teaches that heat-releasable gel preparations are dropped through a molten layer of waxy polymer to form beads with a waxy polymer coating to provide a barrier between water-soluble entrapment means and aqueous solutions within or surrounding the entrapment means. Kosak's entrapment means are degradable only by heat. Furthermore, the apparatus disclosed in Kosak is directed to encapsulating droplets of the reagent in wax shell structure to protect the interior from aqueous solvents.
Therefore, a problem of both Gibbons and Kosak is that they teach encapsulation or entrapment means that are designed to be essentially impervious to aqueous solvents, in which most histo-cytochemical reactions take place. It is not possible to apply Gibbons' or Kosak's method of encapsulation with water soluble encapsulation or entrapment media suspended in aqueous media, since these would disintegrate in aqueous solutions.
Accordingly, it is desirable to provide a method, apparatus and reagents for the automated processing of biological materials for histo-cytochemistry and the like, especially for methods involving ligand pairs, which avoids the need to prepare antisera (or other members of specific ligand pairs) immediately prior to use and which does not require accurate positioning and alignment of the sample substrates.